The stomach epithelium comprises two main compartments with distinct turnover rates and cell compositions termed antrum or pylorus and corpus or main body. While classical mutagenesis experiments suggested the existence of adult stem cells that continuously replenish these cell types, their identity and localization remains elusive. Notably, a recent report identified Lgr5+ stem cells in the antrum, which are capable of multilineage differentiation over long-term and can serve as the cell type of origin for adenomas upon deletion of the tumor suppressor gene Apc. We have recently identified rare Sox2+ cells in the antrum and corpus of mice. Genetic lineage tracing demonstrates that Sox2+ cells can, like antral Lgr5+ cells, give rise to all mature cell types in the stomach for up to 22 months, thus qualifying as bona fide stem cells. These observations raise the following key questions relevant to the biology of stomach turnover and stomach cancer: (1) When in development are Sox2+ stomach stem cells formed; (2) Are Sox2+ stem cells and Lgr5+ stem cells part of the same lineage; (3) What are the molecular and cellular features of Sox2+ stem cells in antrum and corpus; (4) Do Sox2+ stem cells divide symmetrically or asymmetrically and at which rate; (5) Are Sox2+ stem cells responsive to tissue injury and inflammation; (6) Are Sox2+ cells more amenable to tumorigenesis than differentiated stomach cells; and (7) Is Sox2 itself required for stomach development, tissue homeostasis and cancer? We have developed several novel transgenic tools in mice to address each of these fundamental questions in the context of three major aims and multiple subaims. Specifically, we have generated Sox2-GFP reporter mice as well as Sox2-CreER lineage tracing mice to characterize Sox2+ cells at the molecular and cellular levels at different stages of development. Aim 1 entails characterization of the ultrastructure and transcriptome of Sox2+ cells as well as their self-renewal and differentiation kinetics, establishment of an in vitro culture system and evaluation of the lineage relationship between Sox2+ cells and Lgr5+ cells. In Aim 2, we will cross a novel conditional allele for Sox2 to different Cre drivers to assess the requirement for Sox2 at various stages of pre-and postnatal development and in the context of stomach cell injury inflicted by several genetic and chemotoxic models. Given that stomach cancer is the second-most common cause of cancer-related deaths worldwide with relatively little known about its underlying genetic and cellular origins, we propose to test in Aim 3 the susceptibility of Sox2+ stem cells to malignant transformation. Here, we will evaluate whether Sox2+ stem cells are amenable to transformation into adenomas/adenocarcinomas upon deletion of the Apc tumor suppressor and whether Sox2 protein itself is required for tumor formation. Lastly, we will test the hypothesis that the differentiation state of stomach cells influences their amenability to transformation by deleting the E-Cadherin gene, which is mutated in 50% of human diffuse gastric cancer cases, in Sox2+ stem cells, transit-amplifying cells and differentiated chief cells. PUBLIC HEALTH RELEVANCE: Stomach cancer is the second-most common cancer-related cause of death worldwide with an increase seen specifically in diffuse gastric cancer in the United States. Elucidating the fundamental biology of stomach development and homeostasis is imperative for understanding how stomach cancer develops and for identifying cellular and molecular targets for treatment. Thus, by studying Sox2 and Sox2+ stem cells in the normal and malignant stomach, we will gain new basic insights that can be exploited for regenerative medicine as well as for stomach cancer prevention and treatment.